The Mycobacterium tuberculosis ORF Rv0654 encodes a carotenoid oxygenase mediating central and excentric cleavage of conventional and aromatic carotenoids

Article abstract of DOI:10.1111/j.1742-4658.2010.07873.x

Mycobacterium tuberculosis, the causative agent of tuberculosis, is assumed to lack carotenoids, which are widespread pigments fulfilling important functions as radical scavengers and as a source of apocarotenoids. In mammals, the synthesis of apocarotenoids, including retinoic acid, is initiated by the β-carotene cleavage oxygenases I and II catalyzing either a central or an excentric cleavage of β-carotene, respectively. The M. tuberculosis ORF Rv0654 codes for a putative carotenoid oxygenase conserved in other mycobacteria. In the present study, we investigated the corresponding enzyme, here named M. tuberculosis carotenoid cleavage oxygenase (MtCCO). Using heterologously expressed and purified protein, we show that MtCCO converts several carotenoids and apocarotenoids in vitro. Moreover, the identification of the products suggests that, in contrast to other carotenoid oxygenases, MtCCO cleaves the central C15-C15' and an excentric double bond at the C13-C14 position, leading to retinal (C₂₀), β-apo-14'-carotenal (C₂₂) and β-apo-13-carotenone (C₁₈) from β-carotene, as well as the corresponding hydroxylated products from zeaxanthin and lutein. Moreover, the enzyme cleaves also 3,3'-dihydroxy-isorenieratene representing aromatic carotenoids synthesized by other mycobacteria. Quantification of the products from different substrates indicates that the preference for each of the cleavage positions is determined by the hydroxylation and the nature of the ionone ring. The data obtained in the present study reveal MtCCO to be a novel carotenoid oxygenase and indicate that M. tuberculosis may utilize carotenoids from host cells and interfere with their retinoid metabolism. 2010 The Authors Journal compilation

Full text of DOI:10.1111/j.1742-4658.2010.07873.x

The Mycobacterium tuberculosis ORF Rv0654 encodes a
carotenoid oxygenase mediating central and excentric
cleavage of conventional and aromatic carotenoids
Daniel Scherzinger1, Erdmann Scheffer1, Cornelia Bar1, Hansgeorg Ernst2 and Salim Al-Babili1
¨
1 Institute of Biology II, Albert-Ludwigs University of Freiburg, Germany
2 BASF Aktiengesellschaft, Fine Chemicals, and Biocatalysis Research, Ludwigshafen, Germany
Keywords
Mycobacterium tuberculosis, the causative agent of tuberculosis, is assumed
to lack carotenoids, which are widespread pigments fulfilling important
apocarotenoids; carotenoid cleavage
oxygenase; carotenoids; lycopene;
functions as radical scavengers and as a source of apocarotenoids. In mam-
mals, the synthesis of apocarotenoids, including retinoic acid, is initiated
Mycobacterium; retinoids
by the b-carotene cleavage oxygenases I and II catalyzing either a central
Correspondence
or an excentric cleavage of b-carotene, respectively. The M. tuberculosis
S. Al-Babili, Institute for Biology II,
Cell Biology, Albert-Ludwigs University
of Freiburg, Schaenzlestrasse 1, D-79104
Freiburg, Germany
ORF Rv0654 codes for a putative carotenoid oxygenase conserved in other
mycobacteria. In the present study, we investigated the corresponding
enzyme, here named M. tuberculosis carotenoid cleavage oxygenase
(MtCCO). Using heterologously expressed and purified protein, we show
that MtCCO converts several carotenoids and apocarotenoids in vitro.
Moreover, the identification of the products suggests that, in contrast to
other carotenoid oxygenases, MtCCO cleaves the central C15-C15¢ and an
excentric double bond at the C13-C14 position, leading to retinal (C20),
b-apo-14¢-carotenal (C22) and b-apo-13-carotenone (C18) from b-carotene,
as well as the corresponding hydroxylated products from zeaxanthin and
lutein. Moreover, the enzyme cleaves also 3,3¢-dihydroxy-isorenieratene
representing aromatic carotenoids synthesized by other mycobacteria.
Quantification of the products from different substrates indicates that the
preference for each of the cleavage positions is determined by the hydroxyl-
ation and the nature of the ionone ring. The data obtained in the present
study reveal MtCCO to be a novel carotenoid oxygenase and indicate that
M. tuberculosis may utilize carotenoids from host cells and interfere with
their retinoid metabolism.
Fax: +49 761 203 2675
Tel: +49 761 203 8454
E-mail: salim.albabili@biologie.uni-freiburg.de
(Received 19 July 2010, revised 23 August
2010, accepted 8 September 2010)
doi:10.1111/j.1742-4658.2010.07873.x
Introduction
Mycobacterium tuberculosis, the causative agent of
tuberculosis, is an intracellular human parasite infect-
ing approximately two billion people and causing nine
million new cases of tuberculosis and approximately
two million deaths every year worldwide (http://
www.who.int/gtb/). M. tuberculosis cells survive within
the macrophages by preventing the phagosome
maturation, which involves the fusion of phagosomes
with lysosomes, and by avoiding the development of
an appropriate immune response that could activate
the host cell [1–5].
Several mycobacterial species are known to synthe-
size carotenoids [6], a group of isoprenoid pigments
widely distributed in nature and generally composed of
Abbreviations
BCO, b-carotene cleavage oxygenase; MtCCO, Mycobacterium tuberculosis carotenoid cleavage oxygenase.
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D. Scherzinger et al.
A novel carotenoid oxygenase from M. tuberculosis
a C40-polyene. These pigments exert a vital role as
photoprotective pigments and free radical scavengers
and represent essential components of the light-har-
vesting and reaction centre complexes of photosyn-
thetic organisms [7–9]. In animals, carotenoids fulfill
important functions, mainly as precursors of retinoids
[e.g. retinal and vitamin A (retinol)] [10–12]. Retinal
constitutes the visual chromophore of rhodopsins [13],
whereas vitamin A and its derivative retinoic acid are
involved in different processes, such as the immune
response, development and reproduction [14,15].
Retro-retinoids represent a further group of vitamin A
metabolites, including 14-OH-retroretinol and anhydr-
oretinol, which were shown to affect general lympho-
cyte functions such as B-cell and T-cell proliferation
[12,16]. In addition, cleavage products of the acyclic
carotene lycopene (apolycopenals) are considered to
have specific biological activities with respect to several
cellular signalling pathways [17].
oxygenase (BCO) I [24–26] in animals, and CarX and
UmCco1 in the fungi Fusarium fujikuroi [27] and
Ustilago maydis [28], respectively. In addition to BCOI,
mammals contain a second carotenoid cleaving oxy-
genase, BCOII, that mediates the excentric cleavage of
b-carotene at position C9¢-C10¢, leading to the
C13-compound b-ionone and b-apo-10¢-carotenal (C27)
(Fig. 2) [29,30]. The BCO II product b-apo-10¢ carote-
nal may lead to retinoic acid via b-oxidation-like reac-
tions [31].
Several carotenoid oxygenases are known to cleave
apocarotenoids instead of carotenoids [32–34]. For
example, b-apo-10¢-carotenal and several other apoca-
rotenoids (e.g. b-apo-8¢-carotenal and 3-OH-b-apo-10¢-
carotenal) (Fig. 2), represent precursors of retinal and
its derivatives in the cyanobacteria Synechocystis
and Nostoc, converted by the enzymes Synechocystis
A
B
Retinoids belong to the apocarotenoids, a group of
compounds arising through carotenoid cleavage gener-
ally catalyzed by carotenoid cleavage oxygenases,
which are nonheme iron enzymes that target double
bonds in carotenoid backbones, leading to aldehyde or
ketone products [18–21]. However, some members of
this enzyme family act on the interphenyl Ca-Cb dou-
ble bond of lignin [22] and other stilbene-derivatives
such as resveratrol [23]. Retinal is formed through the
symmetrical cleavage of b-carotene at the position
C15-C15¢ (Fig. 1), catalyzed by b-carotene cleavage
C
D
A
B
E
F
G
H
C
Fig. 1. Structure of b-carotene and selected apocarotenoids. The
C
40-polyene of b-carotene (A) constitutes two b-ionone rings. Apoc-
Fig. 2. Cleavage sites and structures of the substrates. The struc-
tures correspond to b-apo-10¢-carotenal (C27; A), 3-OH-b-apo-10¢-car-
otenal (C27; B); b-apo-8¢-carotenal (C30; C); 3-OH-b-apo-8¢-carotenal
(C30; D); b-carotene (E); zeaxanthin (F); lutein (G) and 3,3¢-dihydoxy-
isorenieratene (H). The substrates were cleaved at the C13-C14
and the C15-C15¢ double bonds. Preferred and less targeted sites
are shaded in dark and light gray, respectively. The preference of
the enzyme is deduced from the values presented in Table 2.
arotenoids are designated according to the cleavage site (atom
numbers are depicted) [e.g. oxidative cleavage of the C8¢-C7¢ or the
C13-C14 double bond leads to b-apo-8¢-carotenal (B) or b-apo-13-
carotenone (C), respectively]. Hydroxylation at the C3 ⁄ C3¢ positions
leads to zeaxanthin from b-carotene and to lutein from a-carotene,
an isomer of b-carotene containing one b- and one e-ionone ring.
Aromatic carotenoids (e.g. isorenieratene) contain /-rings (Fig. 2).
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A novel carotenoid oxygenase from M. tuberculosis
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apocarotenoid cleavage oxygenase (formerly named as
Diox1) and Nostoc apocarotenoid cleavage oxygenase
[31,32]. In addition, apo-10¢-carotenal is converted by
the plant carotenoid cleavage dioxygense 8 [34,35] into
the C18-ketone b-apo-13-carotenone (Fig. 1) in the
pathway leading to strigolactones, which act as plant
hormones [36–38] and signalling molecules, attracting
both symbiotic arbuscular mycorrhizal fungi and para-
sitic plants [39,40].
M. tuberculosis is considered to lack carotenoids, in
contrast to the near relative Mycobacterium marinum.
Indeed, the genes required for carotenoid biosynthesis
have disappeared from M. tuberculosis during its evo-
lution, which was accompanied by a reduction of the
genome size [41]. Hence, it is unexpected that the
M. tuberculosis genome H37Rv [42] still contains two
ORFs (i.e. Rv0654 and Rv0913c) coding for putative
carotenoid cleavage oxygenases, indicating the capabil-
ity to convert these pigments. In the present study, we
report the characterization of the Rv0654 encoded
enzyme, which we refer to as the M. tuberculosis carot-
enoid cleavage oxygenase (MtCCO), as suggested by
in vitro and in vivo studies.
Results
MtCCO cleaves apocarotenals at two different
sites
Sequence comparisons suggested that MtCCO is a
member of the carotenoid oxygenase family, showing
approximately 44% similarity to the characterized
enzyme Nostoc carotenoid cleavage dioxygenase [43]
and containing the conserved four histidins residues
required for binding of the cofactor Fe2+ [44]
(Fig. S1). To determine its enzymatic activities,
MtCCO was expressed in Escherichia coli cells as a
glutathione S-transferase fusion protein, and the pro-
tein was purified using glutathione sepharose and
released by the protease Factor Xa (Fig. S2). Using
purified enzyme, we tested the C27-compound b-apo-
10¢-carotenal (Fig. 2) known to be a suitable substrate
for different carotenoid oxygenases [32–34,45]. In addi-
tion, we performed incubations with the stilbene deriv-
ative resveratrol cleaved by some members of the
carotenoid oxygenase family [23], and the isoprenoids
cholecalciferol (vitamin D3), phylloquinone (vitamin
K1) and a-tocopherol, which contain double bonds
that might be targeted by cleavage oxygenases. HPLC
analyses of the in vitro assays did not show any cleav-
age of the noncarotenogenic substrates (data not
shown). By contrast, b-apo-10¢-carotenal was con-
verted into b-apo-13-carotenone (C18) (Fig. 3; I), as
Fig. 3. HPLC analyses of in vitro assays with apocarotenoids. I:
HPLC analyses of the incubation with b-apo-10¢-carotenal (S)
showed the conversion into b-apo-13-carotenone (a; C18) identified
by comparison with the authentic standard (Std). In addition, traces
of retinal (*) were detected. II: The incubation of MtCCO with
3-OH-b-apo-10¢-carotenal (S) led to the formation of 3-OH-b-apo-13-
carotenone (b; C18) and 3-OH-retinal (c; C20). The products were
identical to authentic standards (Stds; b, c) in their UV-visible spec-
tra (insets) and elution characteristics. The chromatogramm
(MtCCO) shows also the formation of a minor product (*).
suggested by comparison with an authentic standard
(Fig. 3; I) and LC-MS analysis (data not shown). This
result indicated the cleavage of the C13-C14 double
bond (Fig. 3). Pointing to the C15-C15¢double bond
as a second, less targeted cleavage site, incubation with
b-apo-10¢-carotenal led also to minor amounts of
b-apo-15-carotenal (retinal; C20) (Fig. 3, I).
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D. Scherzinger et al.
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To determine the effect of b-ionone ring modifica-
tions on the cleavage activity, MtCCO was incubated
with 3-OH-b-apo-10¢-carotenal (Fig. 2). As shown in
the HPLC analysis (Fig. 3, II), 3-OH-b-apo-10¢-carote-
nal was converted into 3-OH-b-apo-13-carotenone
(C18) and 3-OH-b-apo-15-carotenal (3-OH-retinal;
C20), besides a minor product presumably representing
3-OH-b-apo-11-carotenal (C15). The C18 and the C20
products were identified by comparison with authentic
standards (Fig. 3; II) and by LC-MS analyses (data
not shown). These data suggested that MtCCO cleaves
3-OH-b-apo-10¢-carotenal at two different sites, namely
the C13-C14 and the C15-C15¢ double bonds.
In a further approach, MtCCO was incubated with
apocarotenoids of a longer chain length, namely the
C30-compunds b-apo-8¢- and 3-OH-b-apo-8¢-carotenal
(Fig. 2). HPLC analysis (data not shown) of these
incubations revealed the formation of b-apo-13-carote-
none and retinal from b-apo-8¢-carotenal and the cor-
responding hydroxylated derivatives from 3-OH-b-apo-
8¢-carotenal, confirming the cleavage of the C13-C14
and C15-C15¢ double bonds in both substrates. Incu-
bation of apocarotenoids shorter than b-apo-10¢-carot-
enal [i.e. b-apo-12¢- (C25) b-apo-14¢- (C22), b-apo-15¢-
carotenal (retinal; C20) and b-apo-15¢-carotenoic acid
(retinoic acid; C20)] revealed only weak activity with
the C25-compound, whereas substrates with a shorter
chain length were not converted (data not shown).
These results indicate that the b-apocarotenoids
converted by MtCCO must have a chain length of
at least C25.
showed a lower kcat value compared to 3-OH-b-apo-
8¢-carotenal. Although less pronounced,
a similar
tendency was also observed with the C27-compounds.
These data indicated that MtCCO exhibits higher affin-
ities to unsubstituted apocarotenoids but converts their
hydroxylated derivatives faster.
MtCCO mediates a novel cleavage reaction of
C40-carotenoids
To further explore its substrates, purified MtCCO was
incubated with b-carotene under the same conditions
used for in vitro assays with apocarotenoids. However,
only traces of activity were observed in the subsequent
HPLC analysis. Therefore, we applied a higher enzyme
concentration and prolonged incubation times. These
improved conditions resulted in the accumulation of
three different products (Fig. 4, I) identified by their
chromatographic behaviour and LC-MS analyses (data
not shown) as b-apo-13-carotenone (C18), b-apo-15¢-
carotenal (retinal, C20) and b-apo-14¢-carotenal (C22).
This activity demonstrated that MtCCO mediates the
symmetrical cleavage of b-carotene at the C15-C15¢
site, as well as the asymmetrical cleavage of the
C13-C14 or the C13¢-C14¢ double bond.
To test the cleavage of hydroxylated C40-carote-
noids, purified enzyme was incubated with zeaxanthin
and lutein (Fig. 2) under the conditions used for b-car-
otene. As shown in Fig. 4 (II), zeaxanthin was con-
verted to the 3-hydroxylated counterparts of the
products obtained from b-carotene [i.e. 3-OH-b-apo-
13-carotenone (C18), 3-OH-b-apo-15¢-carotenal (3-OH-
retinal, C20) and 3-OH-b-apo-14¢-carotenal (C22)],
which were confirmed by LC-MS analyses (data not
shown). In addition, a minor product was detected,
which may correspond to 3-OH-b-apo-11-carotenal
(C15).
To shed light on the preference of MtCCO with
respect to chain length and hydroxylation of the sub-
strates, kinetic analyses were performed with the b-apo-
8¢- (C30) and b-apo-10¢-carotenal (C27), as well as their
hydroxylated derivatives, 3-OH-b-apo-8¢- and 3-OH-b-
apo-10¢-carotenal. Table 1 gives the Km and kcat values
determined in the biphasic incubation system used; see
also Table S1 and Fig. S3. The lowest Km was obtained
for b-apo-8¢-carotenal, followed by 3-OH-b-apo-8¢-
carotenal and b-apo-10¢-carotenal and, finally, by
3-OH-b-apo-10¢-carotenal. However, b-apo-8¢-carotenal
The composition of the products formed from lutein
was more complicated as a result of the presence of
two different ionone rings (i.e. e and b) (Fig. 2). As
shown in Fig. 4 (III), four major and two minor prod-
ucts were detected in the corresponding HPLC analy-
sis. On the basis of UV-visible spectra and elution
patterns, the two major products, h2 and h1, were iden-
tified as 3-OH-b-apo-15¢-carotenal (3-OH-retinal, C20)
and its almost co-eluting isomer with lower absorption
maximum 3-OH-a-apo-15¢-carotenal, respectively. The
other two major products, g and i, were assumed to be
3-OH-a-apo-13-carotenone (C18) and 3-OH-b-apo-14¢-
carotenal (C22), respectively. This assumption was sup-
ported by the shorter retention time and the lower
UV-visible absorption maximum of product g com-
pared to 3-OH-b-apo-13-carotenone formed from
Table 1. Km and kcat values of MtCCO for different substrates.
Each value represents the mean SD of three independent experi-
ments.
Substrate
kcat (s)1
)
Km (lM)
b-apo-8¢-carotenal
b-apo-10¢-carotenal
3-OH-b-apo-8¢-carotenal
3-OH-b-apo-10¢-carotenal
392.7 0.00
561.7 27.62
1307.6 64.46
764.3 55.25
4.15 0.68
29.36 3.2
21.90 2.6
43.81 5.5
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zeaxanthin (product d; Fig. 4, II). To confirm their
identities, the four major products obtained from
lutein were purified and applied to LC-MS analyses.
As shown in Fig. 5, the products g, h1, h2 and i exhib-
ited the expected molecular ions [M+H]+ of m ⁄z 275,
301, 301 and 327, respectively. The LC-MS analyses
also showed fragments corresponding to the respective
[M+H-H2O]+ ions, which were more abundant in the
analyses of the a- than in those of the b-compounds
(data not shown).
Several mycobacterial species, other than M. tuber-
culosis, accumulate specific carotenoids (i.e carotenoids
with phenolic end groups) [6]. Because MtCCO repre-
sents a subfamily of mycobacterial carotenoid cleavage
oxygenases (Fig. S4), we tested its activity on the aro-
matic carotenoid 3,3¢-dihydroxy-isorenieratene (3,3¢-di-
hydroxy-/, /-carotene) (Fig. 2). As shown in Fig. 4,
IV, this substrate was readily converted into three
major products, j, k, l, besides two minor compounds.
On the basis of their chromatographic properties, we
assumed that the three major products, j, k and l, cor-
respond to 3-OH-u-apo-13-carotenone (C18), 3-OH-/-
apo-15¢-carotenal (C20) and 3-OH-/-apo-14¢-carotenal
(C22), respectively. To confirm this assumption, the
three products were purified and subjected to LC-MS
analyses (Fig. 6), which revealed the expected
[M+H]+ molecular ions of m ⁄ z 271 (product j), 297
(product k) and 323 (product l).
The site preference of MtCCO is determined by
hydroxylation and structure of the ionone ring
In vitro incubations suggested the cleavage of two dif-
ferent sites (i.e. the C15-C15¢ and C13-C14 double
bonds). However, the different amounts of the corre-
sponding products indicated that the two double bonds
are not equally targeted among the substrates tested.
Aiming to determine the enzyme’s preference, the rela-
tive amounts of the C18, C22 and C20 products of three
independent incubations were investigated. The
obtained values (Table 2) indicated that the preference
of the enzyme is highly affected by the presence of the
3-hydroxy-modification in the b-ionone ring. For
example, 80% and 97% of the total product amounts
Fig. 4. HPLC analyses of the incubations of MtCCO with different
carotenoid substrates. UV-visible spectra of the products are
shown in the insets. I: Incubation with b-carotene (B) leading to
b-apo-13-carotenone (a; C18), retinal (b; C20) and b-apo-14-carotenal
(c; C22). II: Incubation with zeaxanthin (Z) showing the formation of
3-OH-b-apo-13-carotenone (d; C18), 3-OH-retinal (e; C20) and 3-OH-
b-apo-14-carotenal (f; C22). III: Incubation with lutein (L) leading to
the supposed products 3-OH-a-apo-13-carotenone (g; C18), 3-OH-a-
apo-15¢-carotenal (h1; C20), its isomer 3-OH-b-apo-15¢-carotenal
(3-OH-retinal; h2) and 3-OH-b-apo-14-carotenal (i; C22). IV: Incuba-
tion with 3,3¢-dihydoxy-isorenieratene (R) showing the formation of
tentative C18- (j), C20- (k) and C22-products (l). In II, III and IV,
traces of other unidentified products (*) were also detected.
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D. Scherzinger et al.
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Fig. 5. LC-MS analyses of the lutein cleavage products. The cleavage products of the incubation with lutein were purified by HPLC and sub-
jected to LC-MS analyses. The products showed the molecular ions [M+H]+ of m ⁄ z 275 (g), m ⁄ z 301 (h1 and h2) and m ⁄ z 327 (i), which are
expected for 3-OH-a-apo-13-carotenone (C18), 3-OH-a-apo-15¢-carotenal (C20), 3-OH-b-apo-15¢-carotenal (C20; 3-OH-retinal) and 3-OH-b-apo-
14¢-carotenal (C22), respectively. The structures of the products are depicted. The spectra of the products with an a-ionone ring exhibited
pronounced [M+H-H2O]+ fragment ions.
Fig. 6. LC-MS analyses of the 3,3¢-dihydroxy-isorenieratene cleavage products. The purified products were subjected to LC-MS analyses and
identified as 3-OH-/-apo-13-carotenone (C18; j), 3-OH-/-apo-15¢-carotenal (C20; k) and 3-OH-/-apo-14¢-carotenal (C22; l), as suggested by the
expected molecular ions [M+H]+ of m ⁄ z 271 (j), m ⁄ z 297 (k) and m ⁄ z 323 (l), respectively. Structures shown correspond to the products.
obtained from b-apo-8¢-b-apo-10¢-carotenal, respec-
tively, were identified as b-apo-13-carotenone (C18)
arising through the C13-C14 cleavage, whereas the
C3-hydroxylated counterparts were mainly targeted at
the C15-C15¢ site, as suggested by the relative higher
amounts of 3-OH-retinal (C20). Similarly, the relative
amounts of the C18 and C22 products resulting from
the cleavage of C13-C14 (or C13¢-C14¢) in b-carotene
were much higher than those of the corresponding
hydroxylated products formed from zeaxanthin. This
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Table 2. Cleavage Specificity of MtCCO. The ratios of products
resulting from the cleavage at the C13-C14 ⁄ C13¢-C14¢ (C18 and C22
)
and at the C15-C15¢ (C20) double bonds are shown, relative to the
total amount of both product types. The values were calculated
from the product peak areas of a MaxPlot 300–550 nm of the
respective HPLC analyses.
C13-C14 ⁄
Substrate
C13¢-C14¢ (%)
C15-C15¢ (%)
b-apo-8¢-carotenal
b-apo-10¢-carotenal
b-carotene
79.6 1.4
97.0 4.6
86.0 13.8
5.0 0.1
20.4 1.3
3.0 0.8
14.0 4.5
95.0 2.3
69.5 1.3
82.9 4.9
54.4 0.2
54.3 3.9
3-OH-b-apo-8¢-carotenal
3-OH-b-apo-10¢-carotenal
Zeaxanthin
30.5 0.6
17.1 8.3
45.6 1.7
45.7 11.5
Fig. 7. Expression of MtCCO in lycopene accumulating E. coli
cells. HPLC analyses of lycopene (L) accumulating E. coli cells
expressing a thioredoxin-MtCCO fusion protein (MtCCO) or thiore-
doxin (Con). The activity of MtCCO resulted in the formation of two
products identified as apo-13-lycopenone (a; C18) and apo-15¢-lyco-
Lutein
3,3¢-dihydoxy-isorenieratene
penal (acycloretinal; b;
C20). The nature of the products was
indicated that the occurrence of the 3-hydroxy-group
favours the symmetrical cleavage at the C15-C15¢ dou-
ble bond. However, this preference is attenuated if the
substrates contain an e- or a /-ionone ring, as deduced
from the incubations with lutein and 3,3¢-dihydroxy-
isorenieratene. Moreover, the asymmetrical cleavage of
lutein appeared to occur only at the C13-C14 site adja-
cent to the e-ionone ring, and not at the C13¢-C14¢ on
the b-ionone site, as indicated by the absence of
b-apo-13-carotenone in the corresponding analyses.
deduced from the UV-visible spectra (insets) and elution patterns.
gov/genomes), with the exception of the extremely
reduced Mycobacterium leprae genome. These enzymes
occur independently of the ecotype and the genome
size (Fig. S4). They are encoded in the 7 Mb genome
of Mycobacterium smegmatis str. MC2 155, in the
reduced 4.4 Mb genome of the intracellular human
parasite M. tuberculosis, as well as in the 6 Mb gen-
ome of Mycobacterium sp. JLS isolated from creosote-
contaminated soil [46]. The number of the carotenoid
oxygenases varies among mycobacterial species, rang-
ing from one in Mycobacterium abscessus to three in
Mycobacterium avium and Mycobacterium vanbaalenii
(Fig. S4). The genome of M. tuberculosis H37Rv con-
tains two genes (Rv0654 and Rv0913c) encoding puta-
tive carotenoid oxygenases. Although the enzymatic
activity of the Rv0913c encoded enzyme remains to be
elucidated, we present data obtained in the present
study (see summary of the substrates analyzed;
Table 3) suggesting that the Rv0654 encoded enzyme
MtCCO is a carotenoid cleavage oxygenase novel with
respect to the cleavage pattern, the conversion of aro-
matic carotenoids and its mycobacterial origin.
MtCCO cleaves lycopene in vivo
In vitro incubations with the acyclic substrate lycopene
did not lead to any detectable conversion, most likely
as a result of the high hydrophobicity hindering solubi-
lization with octyl-b-glucoside used for other sub-
strates. Therefore, we tested the cleavage of lycopene
in vivo. Accordingly, MtCCO was expressed as a thior-
edoxin-fusion in
a
lycopene-accumulating E. coli
strain. Although the decolorization indicated a high
conversion of the substrate, HPLC analyses of the cells
showed only traces of two products (Fig. 7). On the
basis of UV-visible spectra and elution pattern, the
two products were identified as apo-13-lycopenone
(C18; a) and apo-15¢-lycopenal (acycloretinal, C20; b).
These data indicated that MtCCO cleaves carotenoids
in vivo.
The identified cyclic products suggested that MtCCO
can target two different sites in the same substrate (i.e.
the C13-C14 and the C15-C15¢ double bonds). Carot-
enoid oxygenases acting on bicyclic C40-carotenoids
mediate either a central cleavage at the C15-C15¢ dou-
ble bond, leading to two C20-products (e.g. the animal
BCO I [24–26] and the fungal CarX [27]) or an excen-
tric cleavage at a different double bond, which results
in two products that are different in chain length. The
latter reaction was shown for the animal BCO II
Discussion
The biological relevance of carotenoid oxygenases in
mycobacteria is mirrored by their common presence in
the corresponding sequenced genomes available from
the NCBI public database (http://www.ncbi.nlm.nih.
4668
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D. Scherzinger et al.
A novel carotenoid oxygenase from M. tuberculosis
Table 3. Summary of analyzed substrates. +, Cleaved; (+), only
traces of the corresponding C20- and C18-products were observed;
ND, cleavage not detected. Conversion of lycopene was only
detected in vivo.
3,3¢-dihydroxy-isorenieratene by MtCCO, as demon-
strated in the present study, is a novel reaction.
Indeed, MtCCO is the first enzyme shown to cleave
aromatic carotenoids, and this activity may represent
the function of orthologs in mycobacterial species
accumulating these compounds.
Substrate
Cleavage
Cholecalciferol
ND
Many mycobacterial species are known to accumu-
late carotenoids either in a light-independent manner
(scotochromogens) or upon exposure to light (photo-
chromogen) [52]. The synthesis of carotenoids in the
photomorphogenic mycobacterium M. aurum is medi-
ated by a gene cluster consisting of eight ORFs and
organized in two operons [48,53]. Functional charac-
terization of the constituents allowed the elucidation of
the pathway via b-carotene down to isorenieratene
[48], whereas the enzymes responsible for the hydroxyl-
ation leading to 3-monohydroxy- and 3,3¢-dihydroxy-
isorenieratene are still unknown. The enzymes involved
in b-carotene formation are conserved in M. marinum
[54]. On the basis of sequence similarity to the M. mar-
inum phytoene synthase (CrtB) mediating the first
commited step in carotenogenesis, the ORF Rv3397c
encoded enzyme (accession number NP_217914) of
M. tuberculosis H37Rv was identified as a phytoene
synthase homolog [55]. However, sequence compari-
sons (not shown) reveal that this enzyme is rather
related to a S. griseus putative squalene ⁄ phytoene syn-
thase with unknown function (accession number
AAG28701; 60% similarity) than to the authentic phy-
toene synthase from S. griseus (accession number
AAG28701; 43% similarity) or M. marinum (accession
number AAB71428; 39% similarity). This indicates
that the M. tuberculosis H37Rv CrtB-homolog may
catalyze a condensation reaction leading to an isopren-
oid different from phytoene. This is further supported
by the absence of genes coding for other enzymes in
the carotenoid pathway. Taken together, genome anal-
yses exclude a capability of M. tuberculosis to synthe-
size conventional colored carotenoids. However, there
is still the possibility that M. tuberculosis synthesizes
other unknown isoprenoid secondary metabolites,
which may represent the natural MtCCO substrates.
The data reported in the present study suggest that
M. tuberculosis may recruit carotenoids from its host
to produce compounds required for normal growth.
This speculation is supported by the occurrence of
suitable carotenoid-substrates (i.e. b-carotene, lutein,
zeaxanthin and lycopene) in human plasma and tissues
[17]. In addition, the apocarotenoid substrate b-apo-
10¢-carotenal may also be present in lungs, as indicated
by the expression pattern of the corresponding mam-
malian b-carotene cleaving enzyme BCO II [29,30].
Such a scenario would resemble the uptake of other
Phylloquinone
ND
a-tocopherol
Resveratrol
ND
ND
b-apo-8¢-carotenal
b-apo-10¢-carotenal
b-apo-12¢-carotenal
b-apo-14¢-carotenal
b-apo-15¢-carotenal (retinal)
b-apo-15¢-carotenoic acid (retinoic acid)
3-OH-b-apo-8¢-carotenal
3-OH-b-apo-10¢-carotenal
b-carotene
+
+
(+)
ND
ND
ND
+
+
+
Zeaxanthin
+
Lutein
+
3,3¢-dihydoxy-isorenieratene
Lycopene
+
+ (in vivo)
[29,30] and the plant CCD7 [35, 47] enzymes, which
catalyze the cleavage of the C9-C10¢ double bond of b-
carotene leading to b-apo-10¢-carotenal and b-ionone.
The novelty of MtCCO is mirrored by its capability to
act as a central, as well as an excentric cleavage
enzyme. The considerable relative amounts of the cor-
responding products suggested that, at least in the case
of lutein and 3,3¢-dihydroxy-isorenieratene, none of
these two activities is negligible (Table 2).
The expression of MtCCO in E. coli cells accumulat-
ing lycopene indicated
a cleavage of carotenoids
in vivo. However, the amounts of the products ana-
lyzed by HPLC were very low. Similar results were
obtained from b-carotene- and zeaxanthin-accumulat-
ing cells (data not shown). The low cleavage activity in
this in vivo system may be the result of the solubility
of the enzyme, which impedes an access to the carote-
noids accumulated in membranes, as assumed for the
cyanobacterial carotenoid cleavage enzyme Nostoc
carotenoid cleavage dioxygenase, which is localized in
the soluble fraction of Nostoc cells and did not convert
carotenoids in the corresponding accumulating E. coli
strains [43].
The aromatic carotenoid isorenieratene (/,/-caro-
tene; also named leprotene) and its hydroxylated
derivatives are common mycobacterial pigments accu-
mulated in several species [6,48,49]. Isorenieratene
occurs also in some other actinomycetes; for example,
Streptomyces griseus [50] and the coryneform bacte-
rium Brevibacterium linens [51]. The conversion of
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4669

A novel carotenoid oxygenase from M. tuberculosis
D. Scherzinger et al.
overnight treatment with the protease factor Xa in NaCl ⁄ Pi
containing 0.1% Triton X-100 (v ⁄ v) at room temperature.
Purification steps and protein expression were controlled by
SDS ⁄ PAGE.
host lipids (i.e. fatty acids and cholesterol) and their
utilization by this intracellular parasite [56,57]. The
exploitation of the host resources may have allowed
the reduction of the M. tuberculosis genome, by mak-
ing its own biosynthetic capacities dispensable. More-
over, the activities of MtCCO may interfere with the
carotenoid metabolism of the host cell and the pro-
duced retinoids ⁄ apocarotenoids may affect the immune
response. It is striking that the ORF Rv0655 occurring
immediately downstream of the MtCCO gene (Rv0654)
encodes a putative ribonucleotide ABC transporter
ATP-binding protein, which may mediate the transport
of these compounds.
Enzymatic assays
Substrates were purified using thin-layer silica-gel plates
(Merck, Darmstadt, Germany). Plates were developed with
light petroleum ⁄ diethyl ether ⁄ acetone (40 : 10 : 10, v ⁄ v).
Substrates were scraped off in dim daylight and eluted with
acetone. Lutein and zeaxanthin were purified from spinach
and Synechocystis sp. PCC 6803, respectively. Lycopene
and b-carotene were purchased from Roth (Karlsruhe,
Germany). 3,3¢-dihydroxy-isorenieratene was synthesized
according to Martin et al. [58], and apocarotenoids were
kindly provided by BASF (Ludwigshafen, Germany).
Enzyme assays were performed in a total volume of 200 lL
as described previously [34] with some modifications. Some
50 lL of ethanolic substrate solution (200 lm) were mixed
with 50 lL of ethanolic 4% octyl-b-glucoside solution,
dried using a vacuum centrifuge and then resuspended
in 100 lL of 2 · incubation buffer containing 2 mm
Experimental procedures
Plasmid construction
The gene Rv0654 was synthesized by Epoch Biolabs, Inc.
(Missouri City, TX, USA) and cloned into a modified
pBluescript II SK to yield pBSK-Myc1. Rv0654 was then
amplified with the primers MycI-A: 5¢-GGAGGATCCAT
GACCACCGCACAAGC-3¢ and MycI-B: 5¢-GAGCCC
GGGAATTCGACTCACTATAGG-3¢ using one unit of
Phusionꢀ High-Fidelity DNA Polymerase (Finnzymes,
Espo, Finland), in accordance with the manufacturer’s
instructions. The obtained product was purified using
GFXꢀ PCR DNA and Gel Band Purification Kit (Amer-
sham Biosciences, Piscataway, NJ, USA) and cloned into
pBAD ⁄ THIO-TOPOꢁ TA (Invitrogen, Paisley, UK) to
yield pThio-Myc1 encoding MtCCO in fusion with thiore-
doxin. For the expression of glutathione S-transferase
fusion protein, Rv0654 was excised from pThio-Myc1 with
BamHI and SmaI. The fragment was then treated with
T4-DNA polymerase and ligated into SmaI digested and
dephosphorylated pGEX-5X-3 (Amersham Biosciences) to
yield pGEX-5X-Myc1. The identity of the gene was verified
by sequencing.
Tris(2-carboxyethyl)phosphine
hydrochloride,
0.6 mm
FeSO4 and 2 mgÆmL)1 catalase (Sigma, Deisenhofen, Ger-
many) in 200 mm Hepes-NaOH (pH 7.8). Purified MtCCO
was then added to a final concentration of 50 ngÆlL)1 for
apocarotenoid assays or 300 ngÆlL)1 for incubations with
C40-carotenoids, and assays were incubated for 2 and 4 h at
28 ꢂC, respectively. The incubations were stopped by add-
ing one volume of acetone and partitioned twice against
two volumes of light petroleum ⁄ diethyl ether (1 : 4, v ⁄ v).
Lipophilic supernatants were combined, dried and resolved
in chloroform.
In vivo test
Carotenoid-accumulating E. coli TOP10 cells, harbouring
the required biosynthetic genes from Erwinia herbicola,
were transformed with pThio-Myc1 and the void plasmid
pBAD-Thio. Overnight cultures of the obtained strains
were inoculated into LB medium, grown at 28 ꢂC until
D600 of 0.5 was reached and induced with 0.2% arabi-
nose. Cells were then harvested after 4 h and extracted
using acetone ⁄ methanol (7 : 3, v ⁄ v). Extracts were then
dried, resolved in chloroform and subjected to HPLC
analyses.
Protein expression and purification
The plasmid pGEX-5X-Myc1 was transformed into
BL21(TunerꢀDE3) E. coli cells (Novagen, Darmstadt, Ger-
many) harbouring the plasmid pGro7 (Takara Bio Inc.,
Mobitec, Gottingen, Germany), which encodes the groES-
¨
groEL-chaperone system under the control of an arabinose-
inducible promoter. Some 2.5 mL of overnight cultures of
transformed cells were then inoculated into 50 mL of
2 · YT-medium containing arabinose (0.2%, w ⁄ v), grown
at 28 ꢂC until D600 of 0.5 was reached and induced with
0.2 mm isopropyl thio-b-d-glactoside. Cultures were then
grown for 4 h at 28 ꢂC, followed by 12 h at 20 ꢂC. The
fusion protein was purified using glutathione-sepharose 4B
(Amersham Biosciences) and MtCCO was released by
Analytical methods
Substrates were quantified spectrophotometrically at their
individual kmax using extinction coefficients as given by Bar-
ua and Olson [31] or Davies [59]. Protein concentration was
determined using the BioRad protein assay kit (BioRad,
Hercules, CA, USA). A Waters system (Waters GmbH,
4670
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D. Scherzinger et al.
A novel carotenoid oxygenase from M. tuberculosis
Eschborn, Germany) equipped with a photodiode array
detector (model 2996) was employed for HPLC analyses
performed using a YMC-Pack C30-reversed phase column
(250 · 4.6 mm inner diameter, 5 lm; YMC Europe, Scherm-
beck, Germany) with the solvent systems B: metha-
nol ⁄ water ⁄ t-butylmethyl ether (50 : 45 : 5, v ⁄ v) and A:
methanol ⁄ t-butylmethyl ether (500 : 500, v ⁄ v). The column
was developed at a flow rate of 1 mLÆmin)1 with a linear
gradient from 100% B to 43% B within 45 min, to 0% B
within 1 min, then increasing the flow rate to 2 mLÆmin)1
within 1 min and maintaining these final conditions for
another 14 min.
Acknowledgements
This work was supported by the Deutsche Forschungs-
gemeinschaft (DFG) Grants AL892-1-3 and AL892-1-
4, and by a grant to Dr Peter Beyer from the Bill &
Melinda Gates Foundation as part of the Grand Chal-
lenges in Global Health Initiative. We are indebted to
Dr Peter Beyer and Dr Ivan Paponov for valuable
discussions.
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detection of peaks at their individual kmax. The peaks of
the two products were integrated and summed up to 100%.
The relative ratio of each product was determined as the
ratio of the corresponding peak surface.
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Supporting information
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Fig. S2. Coomassie-stained SDS ⁄PAGE gel of MtCCO
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kinetic measurements of recombinant MtCCO cleaving
b-apo-8¢-carotenal.
Fig. S4. Phylogenetic relationship of selected bacterial
members of the carotenoid oxygenase family [Neigh-
bor-joining (NJ) method].
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Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
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should be addressed to the authors.
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